Potential mechanisms for stimulating anaerobic benzene degradation in methanogenic sediments from a petroleum‐contaminated aquifer were evaluated. In short‐term (<2 weeks) incubations, addition of sulfate slightly stimulated benzene degradation and caused a small decrease in the ratio of methane to carbon dioxide production from benzene. However, in longer‐term (>100 days) incubations, sulfate significantly stimulated benzene degradation with a complete shift to carbon dioxide as the end product of benzene degradation. The addition of Fe(III) and humic substances had short‐term and long‐term effects that were similar to the effects of the sulfate amendments. In studies in which anaerobic groundwater was pumped through columns of aquifer sediments, addition of sulfate to the groundwater significantly enhanced the removal of benzene from the groundwater. The stoichiometry of sulfate and benzene removal from the groundwater passing through the sediment columns was consistent with benzene oxidation to carbon dioxide with sulfate serving as the primary electron acceptor. These results demonstrate for the first time that addition of sulfate may be an effective strategy for enhancing anaerobic benzene removal in some petroleum‐contaminated aquifers.